JoVE Encyclopedie van Experimenten
Neurowetenschap
0 weergaven • 4:04 min. • July 8th, 2025
Begin with chemically fixed wild-type and transgenic Drosophila thoraces in a buffer.
In transgenic thoraces, the dorsal longitudinal muscles' neuromuscular junctions contain neurons that express a mutant protein inducing neurodegeneration.
Remove the buffer and flash-freeze the thoraces to prevent tissue damage during bisection.
Add fresh buffer and transfer the thoraces to dissection dishes.
Position a thorax ventral side up and remove the nerve cell clusters to expose the midline.
Bisect along the midline to obtain hemithoraces.
Remove excess tissue.
Incubate the hemithoraces in a blocking buffer to permeabilize the membranes and prevent non-specific antibody binding.
Add fluorophore-conjugated antibodies targeting a glycoprotein expressed in NMJ neurons and muscle actin filaments.
Remove unbound antibodies.
Arrange the hemithoraces on a bridge slide, add mounting media, and mount the tissues.
Visualize under a microscope.
Compared to the wild-type, the mutants show reduced neuronal staining, indicating neurodegeneration, while the muscles exhibit intact fluorescence.
Before beginning the bisections, don cryoprotective gloves and safety glasses and fill a Dewar flask with liquid
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